Enhancing the Expression of CFTR Using Antisense Molecules against MicroRNA miR-145-5p.
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ABSTRACT:
SUBMITTER: Finotti A
PROVIDER: S-EPMC6543725 | biostudies-literature | 2019 Jun
REPOSITORIES: biostudies-literature
Publications
Enhancing the Expression of CFTR Using Antisense Molecules against MicroRNA miR-145-5p.
Finotti Alessia A Gasparello Jessica J Fabbri Enrica E Tamanini Anna A Corradini Roberto R Dechecchi Maria Cristina MC Cabrini Giulio G Gambari Roberto R
American journal of respiratory and critical care medicine 20190601 11
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| S-EPMC6543726 | biostudies-literature
Project description:Peptide nucleic acids (PNAs) are very useful tools for gene regulation at different levels, but in particular in the last years their use for targeting microRNA (anti-miR PNAs) has provided impressive advancements. In this respect, microRNAs related to the repression of cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis, are of great importance in the development of new type of treatments. In this paper we propose the use of an anti-miR PNA for targeting miR-145, a microRNA reported to suppress CFTR expression. Octaarginine-anti-miR PNA conjugates were delivered to Calu-3 cells, exerting sequence dependent targeting of miR-145-5p. This allowed to enhance expression of the miR-145 regulated CFTR gene, analyzed at mRNA (RT-qPCR, Reverse Transcription quantitative Polymerase Chain Reaction) and CFTR protein (Western blotting) level.
| S-EPMC6017273 | biostudies-literature
Project description:Patients with lung adenocarcinoma may benefit from recently developed molecular targeted therapies. However, analogous advanced treatments are not available for patients with lung squamous cell carcinoma (lung SCC). The survival rate of patients with the advanced stage of lung SCC remains poor. Exploration of novel lung SCC oncogenic pathways might lead to new treatment protocols for the disease. Based on this concept, we have identified microRNA- (miRNA) mediated oncogenic pathways in lung SCC. It is well known that miR-145-5p (the guide strand) functions as a tumor suppressor in several types of cancer. However, the impact of miR-145-3p (the passenger strand) on cancer cells is still ambiguous. Expression levels of miR-145-5p and miR-145-3p were markedly reduced in cancer tissues, and ectopic expression of these miRNAs inhibited cancer cell aggressiveness, suggesting that both miR-145-3p as well as miR-145-5p acted as antitumor miRNAs. We identified seven putative target genes (MTDH, EPN3, TPD52, CYP27B1, LMAN1, STAT1 and TXNDC12) that were coordinately regulated by miR-145-5p and miR-145-3p in lung SCC. Among the seven genes, we found that metadherin (MTDH) was a direct target of these miRNAs. Kaplan-Meier survival curves showed that high expression of MTDH predicted reduced survival of lung SCC patients. We investigated pathways downstream from MTDH by using genome-wide gene expression analysis. Our data showed that several anti-apoptosis and pro-proliferation genes were involved in pathways downstream from MTDH in lung SCC. Taken together, both strands of miR-145, miR-145-5p and miR-145-3p are functional and play pivotal roles as antitumor miRNAs in lung SCC.
| S-EPMC5342147 | biostudies-literature
Project description:In microRNA (miRNA) biogenesis, the guide-strand of miRNA integrates into the RNA induced silencing complex (RISC), whereas the passenger-strand is inactivated through degradation. Analysis of our miRNA expression signature of bladder cancer (BC) by deep-sequencing revealed that microRNA (miR)-145-5p (guide-strand) and miR-145-3p (passenger-strand) were significantly downregulated in BC tissues. It is well known that miR-145-5p functions as a tumor suppressor in several types of cancer. However, the impact of miR-145-3p on cancer cells is still ambiguous. The aim of the present study was to investigate the functional significance of miR-145-3p and BC oncogenic pathways and targets regulated by miR-145-5p/miR-145-3p. Ectopic expression of either miR-145-5p or miR-145-3p in BC cells significantly suppressed cancer cell growth, migration and invasion and it also induced apoptosis. The gene encoding ubiquitin-like with PHD and ring finger domains 1 (UHRF1) was a direct target of these miRNAs. Silencing of UHRF1 induced apoptosis and inhibited cancer cell proliferation, migration, and invasion in BC cells. In addition, overexpressed UHRF1 was confirmed in BC clinical specimens, and the high UHRF1 expression group showed a significantly poorer cause specific survival rate in comparison with the low expression group. Taken together, our present data demonstrated that both strands of miR-145 (miR-145-5p: guide-strand and miR-145-3p: passenger-strand) play pivotal roles in BC cells by regulating UHRF1. The identification of the molecular target of a tumor suppressive miRNAs provides novel insights into the potential mechanisms of BC oncogenesis and suggests novel therapeutic strategies.
| S-EPMC5053739 | biostudies-literature
Project description:BackgroundIn recent years, miRNAs found in biological fluids have gained interest as biomarkers of numerous conditions, including migraine. This study aimed to identify differences in the levels of circulating miRNAs in the serum of migraineurs as compared to healthy controls, as well as between patients with different types of migraine and during the ictal and nonictal phases of the condition.MethodsThe screening phase of the study included serum from 13 migraine patients and 13 sex and age matched controls. A panel of 179 miRNAs was analysed using locked nucleic acid SYBR based qPCR. Based on statistical analysis (U Mann-Whitney test) and data from existing literature, nine miRNAs were selected for validation by TaqMan qPCR in an independent cohort of 26 migraineurs and eleven healthy controls. For comparison between the study and control group, U Mann-Whitney test was performed. The differences between patients with chronic and episodic migraine, migraine with and without aura and in ictal and nonictal phases were analysed with Kruskal-Wallis test. The results were corrected for multiple comparisons using Benjamini-Hochberg method. In all analysis p value ≤ 0,05 was considered as significant.ResultsTwo miRNAs, miR-145-5p and miR-26a-5p were significantly upregulated in serum of migraineurs compared to healthy controls. MiRNA-19a-3p was downregulated in patients currently experiencing migraine headache compared to those in the interictal period. No differences were found between patients with different migraine types.ConclusionThe results of our study add to the growing body of evidence for dysregulation of the circulating miRNA profile by migraine. They are further supported by previous reports on differential expression of miR-145-5p, miR-26a-5p and miR-19a-3p in migraineurs. However, more research on larger populations is needed to validate these findings, as well as elucidate the role of circulating miRNAs in the condition. Moreover, to wholly explore the biomarker potential of miRNAs, migraine patients should not only be compared to healthy controls but also to populations with different headache disorders.
| S-EPMC11571994 | biostudies-literature
Project description:Assessing the quality of tissue engineered (TE) cartilage has historically been performed by endpoint measurements including marker gene expression. Until the adoption of promoter-driven reporter constructs capable of quantitative and real time non-destructive expression analysis, temporal gene expression assessments along a timeline could not be performed on TE constructs. We further exploit this technique to utilize microRNA (miRNA or miR) through the use of firefly luciferase reporter (Luc) containing a 3' UTR perfect complementary target sequence to the mature miR-145-5p. We report the development and testing of a firefly luciferase (Luc) reporter responsive to miR-145-5p for longitudinal tracking of miR-145-5p expression throughout MSC chondrogenic differentiation. Plasmid reporter vectors containing a miR-145-5p responsive reporter (Luc reporter with a perfect complementary target sequence to the mature miR-145-5p sequence in the 3'UTR), a Luc reporter driven by a truncated Sox9 (one of the targets of miR-145-5p) promoter, or the Luc backbone (control) vector without a specific miRNA target were transfected into MSCs by electroporation. Transfected MSCs were mixed with untransfected MSC to generate chondrogenic pellets. Pellets were imaged by bioluminescent imaging (BLI) and harvested along a preset time line. The imaging signals from miR-145-5p responsive reporter and Sox9 promoter-driven reporter showed correlated time-courses (measured by BLI and normalized to Luc-control reporter; Spearman r=0.93, p=0.0002) during MSC chondrogenic differentiation. Expression analysis by qRT-PCR suggests an inverse relationship between miR-145-5p and Sox9 gene expression during MSC chondrogenic differentiation. Non-destructive cell-pellet imaging is capable of supplementing histological analyses to characterize TE cartilage. The miR-145-5p responsive reporter is relatively simple to construct and generates a consistent imaging signal responsive to miR-145-5p during MSC chondrogenesis in parallel to certain molecular and cellular events.
| S-EPMC5926818 | biostudies-literature
Project description:Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes, and has been shown to prevent cirrhosis during liver regeneration. Transplantation of mesenchymal stem cells (MSCs) reduces the development of cirrhosis after liver injury. However, the production and secretion of transplanted MSCs in liver were not studied yet. Here we found that the MSCs expressed low levels of HGF protein, but surprisingly high levels of HGF mRNA. Further investigation using bioinformatics analyses and luciferase reporter assay showed that MSCs expressed high levels of microRNA-26a-5p (miR-26a-5p), which targeted 3'-UTR of HGF mRNA to inhibit its protein translation. In vivo, miR-26a-5p-depleted MSCs were transplanted into mice with carbon tetrachloride (CCl4)-induced cirrhosis. We found that suppression of miR-26a-5p in MSCs further ameliorated the severity of liver fibrosis, reduced the portal hypertension and sodium retention, compared to transplantation of control MSCs. Hence, our study suggests that suppression of miR-26a-5p in MSCs may improve their therapeutic effects against cirrhosis through increasing HGF production.
| S-EPMC5376040 | biostudies-literature
Project description:Background. Accumulating evidence indicates that CFTR modulators can be effective for CF sufferers. However, in vivo the microRNAs that regulate CFTR are increased in the CF lung milieu and this can nullify the beneficial effects of CFTR modulators. Methods. A panel of target site blockers (TSBs) designed against the CFTR 3’untranslated region (UTR) were tested for their ability to increase CFTR expression by CFTR 3’UTR luciferase assay and western blot. The effect of two lead TSBs on CFTR activity were tested alone or in combination with selected CFTR modulators in four separate p.Phe508del/p.Phe508del in vitro and ex vivo models (CFBE41o-, Cufi-1, primary bronchial epithelial cells, iPSC-derived lung organoids). Respirable poly-lactic-co-glycolic acid (PLGA) nanoparticles encapsulating the TSBs were formulated and tested. Findings. TSBs that target the miR-145-5p or miR-223-3p binding sites at positons 298-305 and 166-173 in the CFTR 3’UTR, respectively, increased CFTR expression and CFTR anion channel activity, and enhance the effects of Lumacaftor/Ivacaftor or Lumacftor/Tezacaftor in CF BECs. PLGAs encapsulating the TSBs promote CFTR expression in primary BECs and retain their biophysical characteristics following nebulization. Interpretation. Alone or in combination with CFTR modulators, CFTR-targeting TSBs encapsulated in PLGA nanoparticles and nebulized to the lung could overcome microRNA-mediated inhibition of CFTR in CF bronchial epithelium. This strategy represents a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.
2019-03-27 | GSE128861 | GEO
Project description:BackgroundExosomes carrying micro ribonucleic acids (miRNAs) protect against myocardial ischemic injury. In the study, we sought to investigate the protective effect mechanism of M2 macrophage-derived exosome miR-145-5p in hypoxia-reoxygenation (H/R)-induced cardiomyocytes.MethodsM2 macrophages were isolated and induced from blood donated by healthy donors. M2 macrophages were transfected with or without miR-145-5p. Exosomes derived from M2 macrophages were isolated and identified by flow cytometry, nanoparticle tracking analysis, and transmission electron microscopy (TEM). AC16 cells were used to establish an H/R model, and cell activity was detected using a Cell Counting Kit 8 (CCK-8). Western blot was used to detect the expression of gasdermin D (GSDMD), nucleotide-binding domain-like receptor protein 3 (NLRP3), and caspase-1 in the H/R-induced AC16 cells to evaluate pyroptosis. Immunofluorescence staining was used to detect the positive rates of PKH26 and caspase-1. Combined with database prediction, dual luciferase reporter assays were used to validate toll-like receptor 4 (TLR4) as a downstream target molecule of miR-145-5p. A real-time quantitative polymerase chain reaction (RT-qPCR) analysis and western blot were used to detect the expression of TLR4 in the AC16 cells.ResultsFlow cytometry, western blot, nanoparticle tracking and TEM results confirmed the successful isolation of M2 macrophage-derived exosomes. CCK-8 results showed M2 macrophage-derived exosomes decreased the viability of the H/R-induced cells. Western blot results showed the expressions of GSDMD, caspase-1, and NLRP3 were significantly downregulated in the H/R group. Moreover, CCK-8 results showed the M2 macrophage-derived exosome miR-145-5p significantly ameliorated H/R-induced AC16 cellular activity. Western blot results confirmed the expressions of GSDMD, NLRP3, and caspase-1 were significantly downregulated in the macrophage-derived exosome miR-145-5p group compared to the M2 macrophage-derived exosome NC (normal control) group. Immunofluorescence staining results displayed the same trend in terms of the caspase-1 positivity rate. Further, we demonstrated overexpression of TLR4 partially reversed the protective effect of M2 macrophage-derived exosome miR-145-5p in the H/R-induced AC16 cells. Additionally, overexpression of TLR4 reversed the protein expression associated with pyroptosis in M2 macrophage-derived exosome miR-145-5p in the H/R-induced AC16 cells.ConclusionsOur study indicated M2 macrophage-derived exosomes carrying miR-145-5p inhibited H/R-induced cardiomyocyte pyroptosis by downregulating the expression of TLR4.
| S-EPMC9843320 | biostudies-literature
Project description:Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3'UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3'UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content.
| S-EPMC7181265 | biostudies-literature